Revivicor, Inc, Blacksburg, VA 24060, USA.
Xenotransplantation. 2009 Nov-Dec;16(6):477-85. doi: 10.1111/j.1399-3089.2009.00533.x.
Inhibition of the T-cell-mediated immune response is a necessary component of preventing rejection following xenotransplantation with pig alpha1,3-galactosyltransferase gene-knockout (GTKO) organs. Cytotoxic T lymphocyte-associated antigen (CTLA4) is a co-stimulatory molecule that inhibits T-cell activity and may be useful in prolonging graft rejection.
An expression vector was built containing the extracellular coding region of porcine (p) CTLA4 fused to the hinge and CH2/CH3 regions of human IgG1 (pCTLA4-Ig). Pigs transgenic for pCTLA4-Ig, on either a GTKO or wild-type (WT) genetic background, were produced by nuclear transfer and characterized using Western blot analysis, immunofluorescence, ELISA, and necropsy.
Fifteen pCTLA4-Ig-transgenic piglets resulted from five pregnancies produced by nuclear transfer. All transgenic pigs exhibited robust expression of the pCTLA4-Ig protein and most expressed the transgene in all organs analyzed, with significant levels in the blood as well. Despite initial good health, these pigs exhibited diminished humoral immunity, and were susceptible to infection, which could be managed for a limited time with antibiotics.
Viable pigs exhibiting robust and ubiquitous expression of pCTLA4-Ig were produced on both a WT and GTKO background. Expression of pCTLA4-Ig resulted in acute susceptibility to opportunistic pathogens due at least in part to a significantly compromised humoral immune status. As this molecule is known to have immunosuppressive activity, high levels of pCTLA4-Ig expression in the blood, as well as defective development related to exposure to pCTLA4-Ig in utero, may contribute to this reduced immune status. Prophylactic treatment with antibiotics may promote survival of disease-free transgenic pigs to a size optimal for organ procurement for transplantation. Additional genetic modifications and/or tightly regulated expression of pCTLA4Ig may reduce the impact of this transgene on the humoral immune system.
抑制 T 细胞介导的免疫反应是防止用猪α1,3-半乳糖基转移酶基因敲除（GTKO）器官进行异种移植后排斥反应的必要组成部分。细胞毒性 T 淋巴细胞相关抗原（CTLA4）是一种共刺激分子，可抑制 T 细胞活性，可能有助于延长移植物排斥。
构建了一个表达载体，其中包含与人 IgG1 的铰链和 CH2/CH3 区融合的猪（p）CTLA4 的细胞外编码区（pCTLA4-Ig）。通过核转移生产了 GTKO 或野生型（WT）遗传背景下的 pCTLA4-Ig 转基因猪，并通过 Western blot 分析、免疫荧光、ELISA 和尸检进行了表征。
通过核转移产生了五胎十五只 pCTLA4-Ig 转基因仔猪。所有转基因猪均表现出 pCTLA4-Ig 蛋白的强烈表达，并且大多数在分析的所有器官中表达该转基因，在血液中也有显著水平。尽管最初健康状况良好，但这些猪表现出免疫球蛋白减少，易受感染，用抗生素可在有限时间内进行治疗。
在 WT 和 GTKO 背景下均生产出了具有稳健和普遍表达 pCTLA4-Ig 的可行猪。pCTLA4-Ig 的表达导致对机会性病原体的急性易感性，至少部分原因是体液免疫状态显著受损。由于该分子具有免疫抑制活性，因此血液中 pCTLA4-Ig 的高表达以及由于在子宫内暴露于 pCTLA4-Ig 而导致的发育缺陷，可能导致这种免疫状态降低。用抗生素进行预防性治疗可能会促进无病转基因猪的存活，使其达到最佳的器官采购大小以进行移植。对 pCTLA4Ig 的额外遗传修饰和/或严格调控表达可能会降低该转基因对体液免疫系统的影响。
